Dopamine and Octopamine Differentiate between Aversive and Appetitive Olfactory Memories in Drosophila

Appetitive olfactory memory is located in the mushroom bodies

Both forms of odor learning require the mushroom bodies (MBs), a second-order neuropil of the insect olfactory pathway (Heisenberg et al., 1985), and depend on cAMP signaling as revealed by the impairment in mutants such as dunce, rutabaga (rut), and amnesiac (amn) affecting different steps in cAMP metabolism (Tempel et al., 1983). Disruption of the cAMP signaling pathway exclusively in the Kenyon cells of the MBs abolishes olfactory learning (Connolly et al., 1996). On the other hand, MB-specific expression of the wild-type form of the type 1 adenylyl cyclase (AC) rut in an otherwise rut mutant brain rescues performance of olfactory memory (Zars et al., 2000b). Thus, the Kenyon cells of the MBs represent the sufficient set of cells in which cAMP-dependent synaptic plasticity supports memory formation after electric shock reinforcement. To determine whether sugar-dependent memory can also be localized to the MBs, we tested memory performance in genetic mosaics, exclusively restoring rut function within ∼700 Kenyon cells of the MBs (Zars et al., 2000b; Schwaerzel et al., 2002).

Mutant rut²⁰⁸⁰ flies are impaired in sugar memory (37% of wild-type performance) (Fig. 1A,B) and in electric shock memory (42% of wild-type performance) (Fig. 1C) to approximately the same extent. With wild-type rut-cDNA expressed in the MBs (rut²⁰⁸⁰; 247-Gal4/UAS-rut⁺), they show normal sugar and electric shock memory performance (Fig. 1B,C), indicating that formation of sugar and electric shock memories is rut-dependent and located within the same set of ∼700 Kenyon cells (Schwaerzel et al., 2002).

Synaptic output from the same cells is also necessary for memory performance, as shown using the same driver line 247-Gal4 combined with a temperature-sensitive blocker of synaptic transmission [Shibire^ts1, UAS-shi^ts1 (Kitamoto, 2001)]. Both sugar and electric shock learning are normal at the permissive temperature (26°C) in 247-Gal4/UAS-shi^ts1 flies (Fig. 1D,E, white bars) but severely impaired at the restrictive temperature (34°C) (Fig. 1D,E, black bars). The genetic controls do not show any corresponding decrement in performance. In agreement with previous results (Schwaerzel et al., 2002), suppression of electric shock memory at the restrictive temperature is not complete using the UAS-shi^ts1 effector gene (Fig. 1E), whereas sugar memory is abolished completely. This difference between the two learning paradigms might reflect the different signal-to-noise ratios in the two memory scores.

It is a hallmark of electric shock learning that the output of chemical synapses from the Kenyon cells is required only during retrieval but not during acquisition (Dubnau et al., 2001; McGuire et al., 2001; Schwaerzel et al., 2002), suggesting that memory acquisition does not require sustained neural activity in circuits downstream of the MBs or cross talk between Kenyon cells. To test for the role of synaptic output from Kenyon cells in sugar memory, neurotransmitter release was blocked during either training or testing in 247-Gal4/UAS-shi^ts1 flies. Figure 2 shows that blocking Kenyon cell ouptput impairs sugar memory only during retrieval but not during acquisition. This finding indicates that, as with electric shock (Schwaerzel et al., 2002, data not shown), sugar reinforcement (Fig. 2) can modulate the Kenyon cells while their output is turned off (electrical synapses have not yet been reported for Kenyon cells). Together, these experiments show that in electric shock and sugar learning, the olfactory memories are rut-dependent and localized within the same group of 700 Kenyon cells. To further characterize these memories, we investigated the putative modulatory transmitters of the US pathways in electric shock and sugar learning.

Open in a separate window

Figure 2.

Sugar memory formation is independent of synaptic output from Kenyon cells during acquisition. Synaptic output from the Kenyon cells is selectively blocked during either acquisition or retrieval of memory in 247-Gal4/UAS-shi^ts1 flies. A, When 247-Gal4/UAS-shi^ts1 flies are trained at the permissive temperature (26°C) and tested 60 min later at the restrictive temperature (34°C), performance is decreased compared with the heterozygous control groups UAS-shi^ts1/+ and 247-Gal4/+ (p < 0.001). B, In contrast, when 247-Gal4/UAS-shi^ts1 flies are trained at the restrictive and tested at the permissive temperature, memory is not affected compared with the genetic controls (p > 0.05). Temperature is shifted to 34°C 15 min before the training. Means and SEMs of six experiments are shown. The same temperature regimen has been applied to electric shock learning with very similar results (data not shown), confirming previous results (Dubnau et al., 2001; McGuire et al., 2001; Schwaerzel et al., 2002).

Octopamine is required for sugar learning

In the honeybee, OA mediates at least some of the reinforcing capacity of sugar reward in an associative olfactory discrimination task (Hammer and Menzel, 1998; Menzel et al., 1999). Therefore, we investigated the role of OA in sugar and electric shock learning in Drosophila using the TβH-deficient mutant, TβH^M18. The biosynthetic pathway to OA is blocked in this mutant, and it has no detectable levels of OA (Monastirioti et al., 1996). When tested for electric shock memory, mutant TβH^M18 flies performed like the wild-type controls (Fig. 3A), but when tested for sugar memory (Fig. 3B), the mutant was severely impaired, showing PIs near zero. Thus, blocking OA synthesis does not cause a general learning deficit but specifically interferes with sugar learning. This phenotype could be rescued by a transgene containing the wild-type TβH cDNA downstream of the hsp70 promoter. With heat shock, these flies (TβH^M18; hsp70-TβH HS⁺) showed wild-type performance in sugar memory (Fig. 3C). The heat shock itself had no memory-enhancing effect in mutant TβH^M18 flies, supporting the implicit assumption that OA levels can be rescued by restoring enzymatic function.

Open in a separate window

Figure 3.

Octopamine is necessary for the acquisition of sugar memory. A, Flies lacking octopamine caused by a mutation in the mutant TβH^M18 show normal electric shock memory (3 min memory; p > 0.05 compared with control lines described in Materials and Methods; note that electric shock memory of the TβH mutant and control lines is slightly lower than that of our CS wild-type). B, In contrast, no sugar memory is detected in TβH mutant flies (p < 0.001). C, Mutant TβH^M18 flies with a heat-shock inducible TβH⁺ cDNA (TβH; hs1) show normal sugar memory after heat shock (HS⁺) (p < 0.05). Neither the hs1-construct alone (TβH; hs1 - HS^-) nor the heat shock itself have a significant effect compared with the TβH^M18 mutant (p > 0.05) D, Mutant TβH^M18 flies show normal sugar memory after feeding on octopamine (10 mg/ml) for 1 or 18 hr before the experiment (p < 0.001). E, Octopamine is required during acquisition. If TβH^M18 flies are fed octopamine for 1 hr starting right after the training, no memory is detected, although the feeding itself does not abolish 1 hr memory in the control flies (p < 0.001). Data are means and SEMs of six experiments (except for 12 experiments on the TβH^M18 mutant in A and B).

Besides lacking OA, the mutant TβH^M18 accumulates tyramine, its direct precursor (Monastirioti et al., 1996) and a functional neurotransmitter (Nagaya et al., 2002). To test whether the increase in tyramine or the absence of OA causes the phenotype, TβH^M18 mutant flies were fed OA (10 mg/ml) for either 1 or 18 hr before training. OA-fed mutant TβH^M18 flies performed like wild-type flies (Fig. 3D). Surprisingly, a feeding period of as little as 1 hr was sufficient to restore the learning-memory defect, indicating that OA is taken up by the neurons via a rapid mechanism. This is in line with several reports in bees, in which the feeding of OA increases levels of this neurotransmitter in the brain (Schulz and Robinson, 2001; Barron et al., 2002) and behavioral effects occur within the range of minutes after uptake (Pankiw and Page, 2003).

To distinguish between an effect of OA during acquisition and retrieval, OA was fed to the mutant flies just after training, and memory was tested 1 hr later. No rescue of performance was found in these flies, whereas in control flies the same OA feeding regimen had no deleterious effect (Fig. 3E). Therefore, we conclude that OA is required during acquisition. Whether it is also needed during retrieval cannot be decided.

Appetitive and aversive olfactory memories are localized to the same neuropil

Associative behavioral adaptations are mediated by the plasticity of synapses within neural circuits (Kandel, 2001). But what are the smallest units of memory? Do they correspond to the modulation of a single synapse or to the concerted change of many or all synapses in a circuit? Attempts to localize olfactory memory in the Drosophila brain (Heisenberg et al., 1985; de Belle and Heisenberg, 1994; Connolly et al., 1996; Zars et al., 2000a,b; Dubnau et al., 2001; McGuire et al., 2001; Schwaerzel et al., 2002) have provided partial answers to these questions.

In many species, including Aplysia, mouse, and Drosophila, the type-1 AC has been shown to be critical in synaptic plasticity (Lechner and Byrne, 1998; Villacres et al., 1998; Kandel, 2001; Antonov et al., 2003). No cases of cAMP-independent associative synaptic or behavioral plasticity have yet been reported conclusively. In Drosophila, one of the corresponding mutants, rut, shows abnormal performance in every learning paradigm tested so far. By identifying the minimally sufficient set of neurons that in a rut mutant brain need to express a wild-type form of the RUT protein to restore a particular memory performance, one can localize the memory trace of the corresponding behavioral adaptation. This approach was successfully applied to two types of memory in Drosophila, heat box memory (Zars et al., 2000a) and olfactory memory (Zars et al., 2000b).

Using the same approach in a side-by-side comparison between sugar and electric shock reinforcement, our results show that wild-type rut-AC expression in ∼700 Kenyon cells (25-30% of total) rescues memory performance for both kinds of reinforcement. Thus, aversive and appetitive olfactory memories are both mediated by synaptic plasticity in the same group of cells.

Attributing the rescue to an effect on synaptic plasticity in the adult Kenyon cells disregards the possibility that the genetic manipulation might rescue a developmental function of rut-AC, necessary later in the adult for memory. Several lines of evidence argue for an adult function, but only recently has a new genetic manipulation been designed that definitely rules out a developmental effect. Use of a temperature-sensitive Gal80, a suppressor of Gal4, ensured that wild-type rut-AC was expressed only during adulthood (S. E. McGuire, P. T. Le, R. D. Davis, personal communication).

Although our experiments do not specify where in the Kenyon cells cAMP signaling is required, the existing evidence suggests a presynaptic mechanism at Kenyon cell output synapses. At the Drosophila larval neuromuscular junction, cAMP signaling is presynaptically involved in plasticity (Zhong and Wu, 1991). For the sensory-motor synapses mediating classical conditioning of the gill withdrawal reflex in Aplysia (one of the best-studied preparations for this problem), it has been firmly established that the cAMP cascade is involved presynaptically (Antonov et al., 2003). Again, to our knowledge, no conclusive example of a postsynaptic contribution of cAMP signaling has been reported.

In the Aplysia synapses above, plasticity has a postsynaptic component based on a mechanism resembling the NMDA receptor and long-term potentiation in mammals. In Drosophila olfactory conditioning, a similar postsynaptic contribution is unlikely to play a role during the first 3 hr, because this effect would require neurotransmitter release from the presynapse, which can be blocked during acquisition and memory retention without a deleterious effect on memory, using shi^ts1, a conditional blocker of synaptic transmission (McGuire et al., 2001; Dubnau et al., 2001; Schwaerzel et al., 2002).

The tentative presynaptic effect of cAMP signaling locates the synaptic plasticity underlying olfactory memory to the synapses connecting Kenyon cells to MB output neurons. These are found in the MB lobes, including the rostral peduncle and spur (Schürmann, 1987; Yasuyama et al., 2002). Additional support for cAMP signaling to occur in the lobes rather than calyx is derived from the “memory” gene amn, which has been shown to be involved in cAMP regulation (Feany and Quinn, 1995). The putative AMN neuropeptide is required exclusively in two prominent neurons, the so-called dorsal paired medial neurons, that profusely innervate the MB lobes (Waddell et al., 2000). Other components of the cAMP pathway such as rut and receptors for DA and OA [summarized by Crittenden et al., 1998; for dDA1 (Kim et al., 2003)], all are predominantly expressed in the adult MB lobes.

OA and DA differentiate between sugar and electric shock reinforcement

Associative synaptic plasticity depends on the convergence between impulses from two signals, the CS and the US. Considering the proposed role of rut-AC as a molecular coincidence detector (Dudai et al., 1988), one can assume that the MB input neurons carrying the US for sugar and electric shock should also connect to the lobes, although their direct anatomical identification is pending.

Our results show that acquisition of an olfactory memory with electric shock is dependent on the dopaminergic system, whereas acquisition with sugar depends on the octopaminergic system. OA as neurotransmitter in sugar learning seems to be conserved between Drosophila and the honeybee. The bee VUMmx1 neuron, an unpaired neuron localized in the subesophageal ganglion, appears to be octopaminergic and has been shown to carry some of the reinforcing properties of the US. It innervates the calices, antennal lobes, and lateral protocerebrum but not the MB lobes (Hammer and Menzel, 1998). Nevertheless, the learning paradigms used [individual conditioning of the proboscis extension reflex in bees vs the population-based conditioned osmotaxis in Drosophila (Tully, 1986)] are different; therefore, it might be too early to compare the sugar memories in the bee and Drosophila with respect to its organization on a circuit level. Unfortunately, the role of the monoamines in aversive conditioning has not been tested in bees.

These findings raise the question of whether the effects of the two catecholamines on electric shock and sugar learning can be generalized to other appetitive and aversive reinforcers and to positive and negative behavioral modulation in general. In the monkey, midbrain dopaminergic neurons have been described that carry the reinforcing properties of a US in appetitive but not aversive conditioning (Mirenowicz and Schultz, 1996). It will be interesting to see whether a similar dissociation between modulatory systems for appetitive and aversive conditioning, with the contingency between good-bad and monoamines exchanged, also applies to the monkey, and, potentially, to humans.